1. Field of the Invention
The present invention relates to the detection of analytes present in a solvent stream and particularly to the performance of light absorption and fluorescence measurements to determine the chemical properties of small amounts of fluid analyte. More specifically, this invention is directed to analytical cells which are configured as flow-through detectors and especially to microchemical analysis instruments which employ a rigid aqueous liquid core waveguide as a flow cell through which a fluid analyte is directed while being illuminated whereby light absorption by the analyte or fluorescence induced in the analyte may be measured. Accordingly, the general objects of the present invention are to provide novel and improved methods and apparatus of such character.
2. Description of the Prior Art
Ultraviolet (UV) and visible (Vis) light spectrometers, photometers and fluorometers are widely used as detectors in the fields of liquid chromatography (LC), high performance liquid chromatography (HPLC), high performance capillary electrophoresis (HPCE) and capillary electrophoresis (EC). Liquid chromatography applications, for example, are used to separate molecular species owing to their differing flow mobility. The spectrometers associated with chromatography and electrophoresis apparatii detect analytes which result from the separation of the molecular species.
UV/Vis spectrometers have long employed flow cells to detect analytes in liquid chromatography. In the prior art flow cell technology, axial illumination, i.e., illumination along the flow path of the analyte, has been commonly employed. Analytical instrument manufacturers have, for many years, unsuccessfully attempted to overcome two major deficiencies inherent in previously available flow-through spectrometers. A first deficiency relates to increasing utility by reducing analysis cell volume whereby only a small amount of sample fluid is required. The second, and somewhat contradictory problem, has been to increase instrument sensitivity by increasing the light path length to thereby maximize light interaction with the sample fluid. Employing the prior art materials and techniques, it has proven to be exceedingly difficult to increase the light path length without also significantly increasing the volume of the analysis cell. It has also proven very difficult to collimate light into a beam which is sufficiently fine so that the light can travel a long distance through the sample analyte. A long light path-length has, in fact, required resort to the use of a single wavelength collimated source such as a laser. The situation where only a single wavelength light will suffice for all of the measurements required on a given sample is highly unusual. Further, it is difficult and expensive to fabricate a flat window through which light can be externally introduced into a small flow-through analysis cell. In practice, the available light introduction windows have had the result of dispersing, i.e., defocusing, the incident light beam.
To briefly summarize the above, a common application for flow-through cells is the analysis of fluids emerging from chromatographic apparatus wherein analytes have been spatially separated owing to differences in the mobility of molecular species in fluid media. As another application, small volumes of a test analyte are injected into a carrier fluid, usually water, and the carrier fluid is then passed through a flow-through detector cell for analysis by light absorption or induced fluorescent measurements. The sensitivity of prior art optical flow-through detector cells is limited by the ability to ensure that the light path through the analyte is sufficiently long to allow the incident light to interact as fully as possible with the fluid sample. In the prior art this has necessarily implied that the sample volume required should be large and, in many cases, large volume samples are not obtainable. Further, even when a large volume sample is available, and thus a cell having an acceptably long light path length could be constructed, there were significant losses associated with introduction of the analysis light into the cell and the use of non-coherent light was thus generally considered impossible.
A number of techniques for solving the above-discussed long-standing problems have been proposed and in some cases implemented. For example, as shown in published European Patent Application No. 891067001, a flow cell with a light path length of 20 mm could theoretically be achieved through the use of a Z-shaped capillary. This approach, however, was unsuccessful in actual practice because too much of the analysis light, focused directly on one end of the capillary, was lost. High light loss, i.e., low light throughput, limits a flow cell to measurements using a single wavelength light only. Thus, even when the light path length was significantly reduced, Z-shaped capillary flow cells have not proven successful for scanning the full spectrum in the UV and Vis light range.
It should be noted that the use of capillary tubing for a cuvette and glass optical fibers for both illumination and light collection was reported by Vurek and Bowman in 1969, see Analytical Biochemistry, Vol. 29, pgs. 238-247. Such use of glass optical fibers allows the elimination of the window through which light is coupled to the sample and received from the sample. However, mere elimination of the window did not solve the light loss problem since the light travelling through the capillary was not collimated and, accordingly, the maximum path length was limited to about 10 mm.
Efforts to improve on the technique reported by Vurek and Bowman have concentrated on increasing the effective path length of capillary cuvette cells through the use of non-aqueous liquid core waveguide technology. In order for a liquid to function as a light waveguide, the walls of the containment for such liquid, i.e., the walls of the capillary tube, must have a refractive index lower than that of the liquid. Water, the principal solvent for biochemical analytes, has a refractive index of 1.33. Until very recently, there were no materials available which could be used to fabricate a capillary with a refractive index of less than 1.33. In the above-referenced co-pending application, applicant discloses an aqueous liquid filled capillary waveguide.
To continue to discuss the prior art, faced with the inability to employ water as the liquid core of a light waveguide, a number of alternative technologies were proposed. Thus, as reported by K. Fuwa et al in Analytial Chemistry, Vol. 56, pg. 1640, 1984, organic solvents with refractive indices higher than that of the glass wall of a capillary tube were employed to achieve a "long capillary cell" (LCC). Such utilization of organic solvents was reported to achieve an increase in absorbance of about 3.times.10.sup.4 times. This increase in absorbance, however, required a cell 50 meters in length. Futher, the Fuwa et al technique had very limited applicability because only a few liquid organic solvents, such as carbon disulfide and benzine, could be used. A particular disadvantage of using such solvents resides in the fact that their transmittance of UV light is very poor.
Attempts to improve the performance of instruments employing an LCC included coating the internal walls of the capillary with reflective material. Such approaches are discussed by P. Dasgupta in Analytical Chemistry, Vol. 45, page 1401, 1984 and by K. Fujiwara et al in Applied Spectroscopy, Vol. 44, pgs. 1084-1088, 1990. These "mirror" approaches did not prove to be successful since a maximum of about 92% reflection could be obtained. Accordingly, after only 100 wall bounce reflections, only 0.02% of the incident light intensity remained. Dasgupta et al reported nearly a million fold loss of light intensity in a glass capillary cell 10 mm long. Additional problems resulted from the fact that the internally coated silver, which was the favored mirror material, was not flat and would also react chemically with the organic solvents. As an alternative approach, it was suggested that the outer glass surface of an LCC be coated with aluminum to reflect light at the glass-air interface. Such a proposal is discussed by L. Wei et al in Analytical Chemistry, Vol. 55, pg. 951, 1983. As yet another possibility, reported by K. Tsunoda in Analytical Science, Vol. 4, pg. 321, 1988, the capillary glass-air interface was used as the total reflecting surface without any coating. In either case where reflection was from the outer surface of the glass capillary, it has been found that some light would pass many times through the sample, some light would travel further in glass than in the sample and some light would travel only in the glass. Accordingly, the resulting fluid light absorption was not a linear function of analyte concentration and was difficult to predict.
In recent years, it has been proposed to use a fluorocarbon material to define a capillary cell. The use of a fluorocarbon material with a refractive index in the range of 1.38 to 1.5, i.e., a refractive index greater than that of water, is discussed by K. Tusunoda et al in Applied Spectroscopy, Vol. 41, pgs. 163-165, 1990 and J. Taylor et al in the Journal of Chromatography, Vol. 550, pgs. 831-837, 1991. These prior uses of capillary cells comprised of fluorocarbon material required the addition of ethanol and ethylene glycol fluids to increase the refractive index of the sample fluid so as to cause it to be greater than that of the cell defining material.
Thus, after many years of attention by researchers, a basic problem remained, i.e., the necessity of employing organic solvents with refractive indices higher than that of the walls of the capillary cell. It must be noted that, in addition to preventing the use of water as a solvent, the employment of organic solvents results in the measuring instrument being refractive index sensitive. That it, when organic solvents are employed in a LCC utilized for light aborption measurements, the refractive index effect appears on the resulting chromatograms as baseline shifts and false peaks at points where mobile phase composition is changing rapidly. Such rapid changes in mobile phase composition may result during gradient elution and sample injection. Refractive index sensitivity is a serious problem in HPLC detection systems because many organic solvents of many different refractive indices are frequently used. An absorbance detector should not measure refractive index, but should instead measure only absorbance. In conventional prior art flow cells, the lower the refractive index of the carrier fluid, the more light that will be lost. A sudden change in the refractive index of the media in the cell will introduce a sudden change in the light intensity in the detector, giving a false absorbance signal. Many complicated methods have been used with varying degrees of success to reduce this refractive index effect.
It must also be noted that fluorescence detection faces the same problems as absorbance measurement as discussed above. In the case of florescence detection, to enhance sensitivity, the excitation path length should be long. It has been proposed to increase excitation path length through the use of Pyrex glass capillary tubing, having a refractive index of 1.474, and a high refractive index mobile phase (refractive index in the range of 1.5 to 1.6) to form a waveguide. Such a system is described in K. Fujiwara et al, Applied Spectroscopy, Vol. 6, pgs. 1032-1039, 1992. The system described in the referenced article has not proven to be a practical approach because many of the frequently used mobile phases in liquid chromotography have refractive indices lower than 1.474 and, accordingly, will not function as a light waveguide.